Cordotomy-denervation interactions on contractile and myofibrillar properties of fast and slow muscles in the rat

Cordotomy-denervation interactions were studied on contractile and myofibrillar properties of slow (soleus) and fast (extensor digitorum longus) muscles of the rat. The spinal cord was transected midthoracically in neonatal (2-day-old) animals. Two months after birth, a unilateral transection of the sciatic nerve was carried out in both cordotomized and control animals. Five weeks after denervation, contractile properties were tested isometrically in vitro; myofibrillar properties were assessed by histochemical staining of the muscle fibers and by electrophoretic analysis of the myosin heavy chain composition. The following results were obtained: (i) In cordotomized animals the contraction time of the soleus was significantly shorter (-23.3% on average) than that in the control animals and this shortening was accompanied by a proportional slow-to-fast shift in myofibrillar properties. (ii) The extensor digitorum longus properties were not significantly different in the control and cordotomized animals. (iii) Denervation in control animals was followed by a marked increase of contraction and half-relaxation times in the extensor digitorum longus, whereas in the soleus only the half-relaxation time was significantly increased; myofibrillar properties in the soleus showed an appreciable slow-to-fast shift, whereas in the fast muscle the main change was an increase in type 2A fibers to the detriment of type 2B. (iv) In cordotomized animals, denervation caused the soleus contraction time to increase to control values, whereas myofibrillar properties shifted to an even faster pattern; in the extensor digitorum longus denervation caused the same changes seen in the control animals. The results showed that cordotomy at birth caused the soleus to develop as a faster muscle than in the control animals. The concurrent effects of cordotomy and denervation on the myofibrillar properties of the soleus suggest that the slow-to-fast change in these properties is a common consequence of the reduction in the level of motor activity. The opposite effects of the two experimental conditions in the soleus contraction time support the view that the contractile alterations that follow denervation mainly reflect alterations in the muscle activation process.     

Spinal transection and the postnatal differentiation of slow myosin isoenzymes

Newborn rats underwent cordotomy, and the myosin composition of individual muscles was investigated 3 months postoperatively. The results indicate that, after cordotomy, the myosin composition in the extensor digitorum longus and tibialis anterior muscles is normal, whereas in the soleus muscle the myosin has catalytic and molecular properties intermediate between those of adult fast and slow myosin. Together with histochemical data these results indicate that spinal transection causes a developmental arrest in the soleus muscle, at a stage corresponding to a mixed fiber population.     

Long-term effects of spinal cord transection on fast and slow rat skeletal muscle: I. Contractile properties

Contractile properties of rat soleus and extensor digitorum longus muscles were studied 1 year after complete thoracic spinal cord transection (spinal cord level T9). Force-generating capacity and contraction speed were unchanged in the extensor digitorum longus 1 year after transection. However, the rate of contraction and relaxation increased in the soleus as reflected by a decrease in time-to-peak tension and increase in fusion frequency. Additionally, the soleus muscle cross-sectional area decreased significantly (50%) while generating the same absolute tension. Thus, a large increase in soleus specific tension (force per unit area) was observed. These data, in conjunction with the increase in contractile speeds, suggest soleus slow-to-fast fiber type conversion secondary to cordotomy. Discriminant analysis of the contractile properties yields fusion frequency as the best discriminator between muscle groups. Thus, following cordotomy, predominantly slow muscles are affected to a greater extent than fast muscles.     

Effects of hind limb suspension on the development of dystrophic hamster muscle

The effects of hind limb suspension on the development of dystrophic muscles was studied in five dystrophic hamsters (CHF 147, formally UMX 7.1) from age 20 days for 5 months. Their histochemical and contractile properties were compared to five aged-matched controls. Twitch and tetanic tensions (Po) were reduced in treated soleus by 22% and 32% and in plantaris by 29% and 39%, respectively; these reductions were proportional to their smaller cross-sectional areas. Twitch duration and half-relaxation times were not altered in experimental soleus muscle but the mean time to 50% of Po was 19 ms faster than the controls. Experimental soleus and plantaris had higher percentages of type II fibers, i.e., 63% and 97% compared with 37% and 93% in controls, respectively. A similar trend was seen in gastrocnemius and extensor digitorum longus muscles. Areas of both fiber types were reduced in all muscles, but only statistically significant differences in type I areas were seen in soleus and type II areas in the other three muscles. The percentages of fibers with centronuclei in whole cross sections, determined at different positions along the muscle lengths, were reduced by 30% in soleus and extensor digitorum longus. The areas of fiber necrosis were also reduced in experimental extensor digitorum longus and plantaris. These changes in nonweight-bearing muscles support the hypothesis that contractile activity can influence the development of dystrophic properties.     

Effect of denervation on glucose uptake in rat soleus and extensor digitorum longus muscles

From 1 to 14 days after denervation, glucose uptake in the fast extensor digitorum longus and slow soleus muscles in rats was investigated and compared with that of the corresponding intact contralateral muscles. Denervation-induced atrophy in soleus was greater than that in extensor digitorum longus muscle. Glucose uptake in extensor digitorum longus muscle increased significantly, but that in soleus decreased significantly.     

Effect of denervation on cyclic nucleotide metabolism in different types of skeletal muscle of the rat

Slow-twitch soleus and fast-twitch extensor digitorum longus muscles of the rat were denervated unilaterally by sciatic nerve section at mid-thigh level. Activities of adenylate cyclase, guanylate cyclase, low Km and high Km cyclic AMP phosphodiesterase, and cyclic GMP phosphodiesterase were compared on the same, freshly prepared homogenates of denervated and shamoperated contralateral muscles one, two, three, or five days after surgery. As an early consequence of denervation, cyclic AMP metabolism was differentially affected in these different types of skeletal muscle. The adenylate cyclase activity of soleus muscle increased significantly by the second day following denervation and continued to rise through the fifth day, while this enzyme did not increase in denervated extensor digitorum longus even by the fifth day. The high Km cyclic AMP phosphodiesterase was already increased by day one in the denervated soleus, but not until the fifth day in the denervated extensor digitorum longus. Parallel increases beginning the first day were observed for the low Km cyclic AMP phosphodiesterase in both muscles. Since the activity of cytosolic cyclic AMP-dependent protein kinase of soleus muscle was also increased two days following denervation, the changes in cyclic AMP synthetic and degradative enzymes apparently result in a rise in intracellular cyclic AMP concentration. Alterations of the cyclic GMP enzymes following denervation were similar in the soleus and extensor digitorum longus, but were delayed relative to the increases in activity in the cyclic AMP enzymes.     

Periodicities in the rate of on-demand electrical stimulation of the mesencephalic reticular formation to maintain wakefulness.

The effect of chronic administration of corticosteroids on the contractile properties of muscle has not been studied despite the regular occurrence of skeletal muscle weakness and atrophy in steroid myopathy. Two muscles, the predominantly fast-twitch-glycolytic extensor digitorum longus and the predominantly slow-twitch-oxidative soleus muscle of rabbits, were studied to determine if there is a differential effect dependent on the fiber type composition of the muscle. The fast-twitch-glycolytic extensor digitorum longus showed a loss of muscle mass associated with an alteration in contractile properties manifested by slower relaxation. The slow-twitch-oxidative soleus muscle was relatively unaffected by steroid treatment. Isometric contraction in the longus muscle was weaker in the treated animals but showed normal strength when measurements were normalized by comparing contractile data based on the cross-sectional area of the control and treated muscles. The findings suggest the possibility that the alteration in contractile properties could be related to changes in myosin or other contractile protein components of the fast-twitch-glycolytic extensor digitorum longus muscle. Changes in sarcoplasmic reticulum, although less likely, are not excluded by the present data.     

Appearance in slow muscule sarcolemma of specializations characteristic of fast muscle after reinnervation by a fast muscle nerve.

We utilized quantitative freeze-fracture electron microscopy to study the plasticity of orthogonal clusters of 60-Å particles (the “square array”) found in the sarcolemma of fast-twitch muscle. The membrane macromolecular composition of normally slow-twitch rat soleus muscle was examined 1 year after surgical reinnervation by the nerve from fast-twitch extensor digitorum longus muscles. The isometric contraction times and histochemical profiles were monitored and it was confirmed that conversion of fiber types had occurred. The sarcolemma of the switched “fast” soleus developed square arrays of 60-Å particles characteristic of fast-twitch muscle whereas the sarcolemma of the contralateral control, the “slow” soleus, contained only random particles. Square array density per square micrometer in cross-reinnervated fast soleus fibers resembled that of normal fast extensor digitorum longus muscles and varied as a function of distance from the neuromuscular junction. This experiment demonstrates that the appearance of these unusual clusters of 60-Å particles is neuronally regulated. We further suggest that these macromolecules are under the influence of the same subtle aspects of innervation that regulate the differentiation of myosin adenosine triphosphatase and thereby the contractile behaviors of fast- and slow-twitch muscle. The function of the sarcolemmal square array is unknown; however, a correlation with a membrane property that is more highly developed in fast-twitch muscle is to be expected.     

Caffeine contractures in denervated slow-twitch and fast-twitch muscles of the rat

Caffeine contractures (25 mM) and twitches were registered in vitro (34 degrees C) in normal and denervated soleus and extensor digitorum longus of the rat. Both muscles lost weight progressively after denervation although the loss of weight in soleus muscles was more manifest. Denervated extensor digitorum longus muscles showed an increase in the caffeine contracture tension (expressed in g/g muscle) whereas the response generated by denervated soleus muscles was smaller than the control values. The time to peak of the caffeine responses was shortened in both types of muscles after denervation. Twitches were larger and had a slower time course than in normal muscles, especially in the extensor digitorum longus muscles. These findings suggest that some changes in the excitation-contraction coupling could be responsible for the potentiation of the twitches in denervated muscles.     

Colchicine-induced differential sprouting of the endplates on fast and slow muscle fibers in rat extensor digitorum longus, soleus and tibialis anterior muscles

The patterns of sprouting of motor endplates were examined in fast extensor digitorum longus and slow soleus muscles and in tibialis anterior muscles containing fast and slow muscle fiber types. A histochemical technique combining nerve silver impregnation and endplate cholinesterase staining was developed for this task. Temporal examination of the innervation was conducted 3, 7 and 10 days after either a 45 or 90 min application of the ipsilateral sciatic nerve with 5 mM colchicine. This dosage of drug did not cause detectable axon or muscle fiber degeneration, unlike 60 mM which was highly neurotoxic. At 3 days following treatment with the lower concentration, there were no significant differences in the percentages of intranodal, preterminal and ultraterminal sprouts between the normal (non-treated), sham-treated, contralateral systemic-control and drug-treated groups of muscles. By 7 and 10 days, the muscles on the drug-treated side exhibited significant increases in the 3 types of sprouts. Collateral sprouting was uncommon: most outgrowths remained on the muscle fibers innervated by the parent axons. Endplates in the tibialis anterior muscles of the control and drug-treated groups were classified Complex, Intermediate or Simple according to the relative degrees of branching of the terminal arbors. The occurrence of endplate classes and muscle fiber types was correlated in the superficial and deep regions of this muscle. Complex endplates innervated fast glycolytic fibers, Intermediate endplates supplied fast oxidative glycolytic fibers, and Simple endplates served slow oxidative fibers. In response to colchicine, the endplates of the slow muscles sprouted more than those of fast muscles while the innervation of slow fiber types sprouted less than that of fast fiber types. Furthermore, intranodal sprouts were more prevalent in slow muscles and ultraterminal sprouts more numerous in fast muscles whereas intranodal sprouts predominated on fast fiber types and ultraterminal sprouts were characteristic of slow fiber types. These apparently contradictory results were reconciled when it was noted that soleus endplates were mostly Complex and Intermediate, and the extensor digitorum longus contained more Simple endplates. Thus, consistency of sprouting patterns among endplate types of the 3 muscles was recognized when the pre-existing branching patterns were considered. This indicated that the patterns of sprouting were determined by the motor neurons rather than the muscle fibers. The observed sprouting responses supported the hypothesis that colchicine treatment of motor axons caused muscle fibers to elaborate a diffusible sprout-inducing factor.     

Muscular abnormalities in experimental hypothyroidism of rats with special reference to the mounding phenomenon

The mounding phenomenon was examined in vitro on the excised extensor digitorum longus or soleus muscle of hypothyroid rats. The mounding phenomenon was more prominent and lasted longer in hypothyroid muscles than in controls. In single skinned muscle fibers of both type 1 and type 2 from hypothyroid animals, the maximum tension produced by Ca ion was reduced and activity of Ca uptake by the sarcoplasmic reticulum was also decreased, judging from the response to caffeine. Decreased Ca uptake might retard the relaxation of contracture, thereby rendering the mounding more prominent. Morphological studies of both extensor digitorum longus and soleus muscles showed an increase of type 1 muscle fiber population as well as mild changes in arrangement of the internal membrane system. However, there was no muscle necrosis or other derangements. These results supported the idea that functional abnormality of the sarcoplasmic reticulum could be the basis of the exaggeration of the mounding phenomenon in hypothyroidism.     

Potassium and caffeine contractures in limb muscles of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice

The strength of contractures, produced by 15 to 146 mM [K]0 (as L-glutamate), was measured in isolated small bundles of muscle fibers from the fast-twitch extensor digitorum longus and from the slow-twitch soleus of normal and dystrophic (C57 BL/6J dy2J/dy2J) mice. The analysis of the relation between the maximal amplitude of the contracture vs the membrane potential and the time constant of relaxation of the K-contractures has shown that dystrophy induced an attenuation of the differences between fast- and slow-twitch muscles. The repriming of K-contractures was more affected by changes in [Ca]0 in normal soleus than in normal extensor digitorum longus and this difference was unaffected by dystrophy. For both types of muscles, the ability of caffeine to produce contractures was reduced in dystrophic muscle and this modification was not related to a change in the fiber typing.     

Temperature effects on the kinetics of force generation in normal and dystrophic mouse muscles

The kinetics of isolated extensor digitorum longus and soleus muscles from normal and genetically dystrophic (129/ReJ dy/dy) mice were studied at temperatures from 8 to 38 degrees C. The rate constants for the exponential rise of tetanic force and for the exponential decay of force during an isometric twitch or short tetanus were similar in normal and dystrophic soleus muscles, but the decay rates were significantly reduced in dystrophic extensor digitorum longus muscles. The temperature dependence for several rate constants for isometric twitches and tetani was similar in all muscles studied, suggesting that the same rate limiting processes apply to fast and slow, normal and dystrophic muscles. Thus, the contractile proteins and those in the sarcoplasmic reticulum of dystrophic muscle are probably normal. The slower relaxation phase in dystrophic extensor digitorum longus muscles is compatible with a reduction in Ca2+-pumping sites in the sarcoplasmic reticulum, perhaps secondary to a change in motor unit composition. Some changes in the temperature dependence for measured times, toward those of soleus muscles, is consistent with the increased proportion of slow twitch motor units in dystrophic extensor digitorum longus muscles.     

Fetal-type creatine kinase in rat fast and slow muscles during denervation and reinnervation

Creatine kinase (CK) has three forms of isozymes; CK-BB, CK-MB, and CK-MM. In adult rats they show a specific tissue distribution: the BB form in the brain, the MB form in the heart, and the MM form in skeletal muscle. In embryonic skeletal muscles only the BB and MB forms are found. Adult slow-twitch muscles contain more fetal type creatine kinase (CK-B) than do fast-twitch muscles. In the present experiment the effect of denervation and reinnervation on the CK-B concentration was investigated in rat fast (extensor digitorum longus)- and slow (soleus)-twitch muscles by a highly sensitive immunoassay. Denervation of these muscles produced a progressive increase in CK-B concentration in both muscles. When the sciatic nerve was cut and immediately sutured, the CK-B concentration in both muscles showed a gradual reduction after an initial increase. By the 34th postoperative week the CK-B concentration in the soleus was about one-half that of the contralateral control, whereas that in the extensor digitorum longus was nearly normal. After cross union of the nerves innervating the muscles, the CK-B concentration in the soleus was reduced at 35 weeks to about one-half normal, but that in the extensor digitorum longus was always higher than the control value. After self-reunion of the nerves, the CK-B concentration at the 20th week was approximately normal in the extensor digitorum longus and significantly increased in the soleus. We suggest that the motoneurons normally innervating the extensor digitorum longus have a greater capability in suppressing the production of CK-B than do the soleus motoneurons.     

Factors affecting muscle fiber transformation in cross. Reinnervated muscle

Nerves of two fast muscles [peroneus longus (PL) and extensor digitorum longus (EDL)], having different type 2 muscle fiber compositions, were used to cross-reinnervate the slow soleus muscle in the rat. Contraction characteristics, histochemical muscle fiber type compsotions and myosin heavy chain (MHC) isoform compositions were determined for the reinnervated muscles. Shortening velocity increased in soleus muscles crossreinnervated with EDL nerve [X-SOL(EDL)] but not in muscles cross-reinnervated with PL nerve [X-SOL(PL)]. Type 2A MHC isoform content was increased in X-SOL(EDL) but not in X-SOL(PL), where MHC isoform composition remained similar to normal soleus. The complement of type 1 (slow) muscle fibers was reduced and that of type 2 (fast) fibers increased in both types of X-SOL muscle, but this change was significantly greater in X-SOL(EDL); the majority of the type 2 fibers in X-SOL muscles were of type 2A. Results show that “the type 2 composition” of the reinnervating motoneuron pool is an important factor in determining the transformation of a target slow muscle after cross-reinnervation. © 1993 John Wiley & Sons, Inc.     

Localization of the pool of G4 acetylcholinesterase characterizing fast muscles and its alteration in murine muscular dystrophy

The distributions of acetylcholinesterase and its molecular forms within muscles of normal and dystrophic 129/ReJ mice were established by a concomitant cytochemical and biochemical study performed on 1-mm serial sections of three predominantly fast muscles, i.e., anterior tibialis, extensor digitorum longus, and sternomastoid, as well as the slow-twitch soleus. This comparative study showed the following main findings. 1) In every muscle of both normal and dystrophic mice a) the three asymmetric forms were confined to the motor zone where they systematically codistributed with the endplates, and b) all globular forms, including G4, were concentrated at the motor zone from which they extended over the entire muscle length along a concentration gradient. 2) In the normal muscles, the perijunctional sarcoplasmic cytochemical reaction exhibited by individual fibers was grouped into a well-defined cojunctional acetylcholinesterase compartment in which the endplates were embedded. The overall intensity of the cojunctional cytochemical reaction was either high or low according to whether the muscle was predominantly fast or slow. 3) This cojunctional acetylcholinesterase compartment varied in close parallelism with G4 and thus appeared as the cytochemical correlate of the G4 molecules concentrated around the endplates. In particular, as the shape of the motor zone progressively increased in complexity along with the intricacy of the muscle fiber organization, from sternomastoid to extensor digitorum longus to anterior tibialis, so did both the relative volume occupied by the cojunctional acetylcholinesterase compartment and the proportion of G4. 4) The motor zone of the normal fast-twitch muscles characteristically differed from that of the soleus by the presence of a G4-rich environment around the endplates, which was cooperatively provided by the surrounding fibers. 5) In dystrophic muscles, this cojunctional G4-rich compartment was lost: the cojunctional cytochemical compartment was no longer discernable, while G4 was reduced to a minimal low level similar to that characteristic of the normal soleus.     

Changes in sarcomere length following tenotomy in the rat

The medial belly of the gastrocnemius and the extensor digitorum longus muscles of rats were tenotomized. One day following tenotomy, the mean sarcomere length of the fast medial gastrocnemius was 1.8 microns, a value comparable to that of tenotomized slow soleus. The mean sarcomere length of the tenotomized extensor digitorum longus, however, was 2.0 microns, a figure which differed significantly from the values obtained for both the soleus and the gastrocnemius. Histological preparations showed the presence of central core degeneration in slow fatigue-resistant fibers of the tenotomized gastrocnemius comparable to that seen in the soleus. No changes were found in the fibers of the tenotomized extensor digitorum longus. The fact that central core lesions were produced in the fibers of soleus and medial gastrocnemius but not in the extensor digitorum longus may be related to the lesser reduction in sarcomere length following tenotomy of the latter muscle.     

Protein synthesis by ribosomes from normal and denervated red and white muscle

No difference was found in the efficiency for protein synthesis of isolated ribosomes from the soleus (red) and extensor digitorum longus (white) muscles of the rat. During the first few days after denervation there was a decrease in the efficiency of ribosomes from the soleus but not from the extensor digitorum longus. This could account for the more rapid atrophy of the former muscle compared with that of the extensor digitorum longus.     

A fractal characterization of the type II fiber distribution in the extensor digitorum longus and soleus muscles of the adult rat

A method is proposed for the quantitative characterization of fiber type spatial distribution by means of the (correlation) fractal dimension. The method is applied to type II fiber distributions of the soleus and extensor digitorum longus muscles of the adult rat. The results obtained suggest that these distributions have fractal properties with a strong tendency for spreading, more pronounced in soleus muscle. The density of muscle fibers or the age do not seem to alter the features of the distribution. Computer-generated random patterns have virtually the same fractal dimension as the extensor digitorum longus distributions but fail to approximate those of the soleus muscle. This fractal method could find application as an alternative in the quantitative assessment of the fiber type grouping. © 1995 John Wiley & Sons, Inc.     

Hypogravity-induced atrophy of rat soleus and extensor digitorum longus muscles

Prolonged exposure of humans to hypogravity causes weakening of their skeletal muscles. This problem was studied in rats exposed to hypogravity for 7 days aboard Spacelab 3. Hindlimb muscles were harvested 12-16 hours postflight for histochemical, biochemical, and ultrastructural analyses. The majority of the soleus and extensor digitorum longus fibers exhibited simple cell shrinkage. However, approximately 1% of the fibers in flight soleus muscles appeared necrotic. Flight muscle fibers showed increased glycogen, lower subsarcolemmal staining for mitochondrial enzymes, and fewer subsarcolemmal mitochondria. During atrophy, myofibrils were eroded by multiple focal losses of myofilaments; lysosomal autophagy was not evident. Tripeptidylaminopeptidase and calcium-activated protease activities of flight soleus fibers were significantly increased, implying a role in myofibril breakdown. Simple fiber atrophy appears to account for muscle weakening during spaceflight, but fiber necrosis is also a contributing factor.